In one aspect, the present invention relates to methods, reagents and support surfaces for use in solid phase (e.g., repetitive or combinatorial) synthesis. In another aspect, the invention relates to substituted polyacrylamide reagents. In yet another aspect, the invention relates to reagents for use in modifying support surfaces, and in particular, the use of photochemical means to attach such reagents to such surfaces.
Solid phase synthesis has evolved tremendously since the seminal work of R. B. Merrifield in 1963. Typically, the reactions used are the same as ordinary synthesis, with one of the reactants being anchored onto a solid support. Solid phase synthesis can be used, for instance, for the synthesis of polynucleotides, polysaccharides, and polypeptides, as well as other applications in repetitive syntheses and combinatorial chemistry.
The basic advantage of the solid phase technique is that the support (including all reagents attached to it) remains insoluble and is therefore easily separated from all other reagents. Excess reagents, other reaction products and side products, are quickly and efficiently removed upon removal of the solvents. Purification of the solid phase species is rapid and complete as well, and the entire process can be automated.
In recent years, the principles of solid phase synthesis have been applied to a new methodology known as xe2x80x9ccombinatorial chemistryxe2x80x9d. Scientists use combinatorial chemistry to create large populations of molecules, or libraries, that can be screened efficiently en masse. By producing larger, more diverse compound libraries, companies increase the probability that they will find novel compounds of significant therapeutic and commercial value. The field represents a convergence of chemistry and biology, made possible by fundamental advances in miniaturization, robotics, and receptor development.
As with traditional drug design, combinatorial chemistry relies on organic synthesis methodologies. The difference is the scopexe2x80x94instead of synthesizing a single compound, combinatorial chemistry exploits automation and miniaturization to synthesize large libraries of compounds. But because large libraries do not produce active compounds independently, scientists also need to find the active components within these enormous populations. Thus, combinatorial organic synthesis is not random, but systematic and repetitive, using sets of chemical xe2x80x9cbuilding blocksxe2x80x9d to form a diverse set of molecular entities.
There are at least three common approaches to combinatorial organic synthesis. During arrayed, spatially addressable synthesis, building blocks are reacted systematically in individual reaction wells or positions to form separate xe2x80x9cdiscrete molecules.xe2x80x9d Active compounds are identified by their location on the grid. This method has been applied in scale (as in the Parke-Davis Pharmaceutical xe2x80x9cDIVERSOMERxe2x80x9d technique), as well as in miniature (as in the Affymax xe2x80x9cVLSIPSxe2x80x9d technique). The second technique, known as encoded mixture synthesis, uses nucleotide, peptide, or other types of more inert chemical tags to identify each compound.
During deconvolution, the third approach, a series of compound mixtures is synthesized combinatorially, each time fixing some specific structural feature. Each mixture is assayed as a mixture and the most active combination is pursued. Further rounds systematically fix other structural features until a manageable number of discrete structures can be synthesized and screened. Scientists working with peptides, for example, can use deconvolution to optimize, or locate, the most active peptide sequence from millions of possibilities.
On a related subject, surfaces modified to provide reactive groups or other desired functionalities have long been used for performing solid phase syntheses of both polymeric and nonpolymeric molecules. A variety of solid phase resins are commercially available, e.g., those available from Argonaut Technologies, including their line of Polystyrene, ArgoGel(trademark), and ArgoPore(trademark) resins. Along similar lines, published International Patent Application No. WO9727226 (xe2x80x9cHighly Functionalized Polyethylene Glycol Grafted Polystyrene Supportsxe2x80x9d), assigned to Argonaut Technologies, describes polymers and graft copolymers having a backbone of poly(methylsytrene) and side chain polymers of poly(ethylene oxide).
Such supports are also described in JW Labadie (xe2x80x9cPolymeric Supports for Solid Phase Synthesisxe2x80x9d), Current Opinions in Chemical Biology 2:346 (1998). This article describes, for instance, the manner in which functional groups can be introduced into lightly cross-linked polystyrene, using either functional styrene monomers or in a post-functionalization step. In both approaches, however, the functional groups are apparently attached to the polystyrene polymers used to form the support (e.g., bead) itself, e.g., as opposed to being added as a separate coating to a pre-existing support.
The Labadie article also describes the use of PEG-grafted polystyrene, e.g., in the form of the xe2x80x9cTentaGelxe2x80x9d product prepared by grafting ethylene oxide to hydroxyl-functional polystyrene. The article further describes the manner in which various xe2x80x9cshortcomingsxe2x80x9d associated with PEG grafts resins have been overcome by a graft resin identified as xe2x80x9cArgoGel(trademark)xe2x80x9d which is designed with a bifurcation at the polystyrene-graft linkage through the use of a polystyrene diol as the base resin. With each of these approaches, the resultant polymers appear to be limited to functional groups at their terminal ends, as opposed to having functional groups in multiple positions along the length of the polymers.
On yet another subject, a variety of polymeric compositions have been described for use as electrophoretic gels. See generally, Righetti, et al., J. Chromatog. B. Biomed. Sci. 10;699(1-2):63-75 (1997) which describes recent advances in polyacrylamide gel electrophoresis.
See, for instance, U.S. Pat. No. 5,470,916, for xe2x80x9cFormulations for Polyacrylamide Matrices in Electrokinetic and Chromatographic Methodologiesxe2x80x9d. The ""916 patent describes formulations obtained via polymerization or co-polymerization of a unique class of monomers.
See also, U.S. Pat. No. 5,785,832, for xe2x80x9cCovalently Cross-linked, Mixed-bed Agarose-polyacrylamide Matrices for Electrophoresis and Chromatographyxe2x80x9d, which describes polyacrylamide matrices based on a novel class of N-mono- and di-substituted acrylamide monomers. The ""832 patent describes the manner in which mixed-bed matrices of the type polyacrylamide-agarose, covalently linked (cross-linked), are useful in the separation of fragments of nucleic acid, particular DNA, of intermediate size (from 50 to 5,000 base pairs) and of high molecular mass proteins ( greater than 500,000 Da). The ""832 patent provides covalently-linked polyacrylamide-agarose mixed-bed matrices suitable for use in the separation of fragments of nucleic acids of intermediate size.
Substituted polyacrylamides such as those described above have been restricted to use in preparing electrophoretic gels, and, to the best of Applicant""s awareness, have not previously been attached to surfaces, let alone attached for the purpose of providing a solid phase synthetic surface, or by photochemical means.
On a separate subject, the assignee of the present invention has previously described the modification of surfaces for a variety of purposes, and using a variety of reagents. In particular, these reagents generally involve the use of photochemistry, and in particular, photoreactive groups, e.g., for attaching polymers and other molecules to support surfaces. See, for instance, U.S. Pat. Nos. 4,722,906, 4,979,959, 5,217,492, 5,512,329, 5,563,056, 5,637,460, 5,714,360, 5,741,551, 5,744,515, 5,783,502, 5,858,653, and 5,942,555.
The present invention provides a method for performing solid phase synthesis, the method comprising the steps of:
a) providing a support material providing a surface adapted for use in solid phase synthesis,
b) providing a polymeric reagent formed by the polymerization of monomers of the formula: 
wherein R1 represents hydrogen or C1-C6 alkyls, and wherein R2 and R3, independently among them, represent hydrogen, C1-C6 alkyls or phenyls containing one or more reactive substituents selected from 
OR5, or SR5 (where R4 is a C1-C6 alkyl or a heterocyclic ring containing one or more nitrogen atoms and R5 is a C1-C6 alkyl or phenyl containing one or more reactive substituents selected from 
c) applying the reagent to the support surface and covalently attaching the polymeric reagent to the support surface,
d) providing a first reactive monomer adapted for solid phase synthesis, e.g., selected from nucleotides and amino acids, the monomer comprising a corresponding group thermochemically reactive with the bound reactive substituent, and preferably also comprising one or more groups reactive with a subsequent, second monomer unit in the course of solid phase synthesis,
e) contacting and reacting the first monomer with the polymeric reagent upon the support surface under conditions suitable to react the corresponding group with the bound reactive substituent, thus providing a growing polymeric chain, and
f) providing and sequentially attaching subsequent monomers to the growing polymeric chain to obtain a desired polymeric product.
Once formed in this manner, the resultant polymeric product can either be retained and used in situ (e.g., in its bound condition), or it can be cleaved and removed from its position upon the support, in order to be used in a different manner.
In another aspect, the present invention provides a polymeric reagent composition adapted to be coated onto a support surface in order to provide that surface with a high density of reactive groups. The surface, thus coated, can be used for any suitable purpose, and is particularly well suited for use as a solid phase synthesis support surface. The synthesis support surface, in turn, can be used in repetitive and combinatorial syntheses such as the synthesis of polynucleotides, polypeptides and polysaccharides. The polymeric coating can be used to provide increased effective surface area, particularly in situations in which the effective area of the support surface is itself limited, as on the surface of a bead or silicon wafer. In so doing, the polymeric coating provides an optimal combination of such properties as reactive group density and surface area or volume.
In a preferred embodiment, the polymer reagent is provided in the form of a hydrophilic or amphiphilic polymeric reagent adapted to be coated onto a support surface via stable covalent bonds in order to provide the surface with a high, but controllable, density of functional groups suitable for solid phase synthesis of peptides, oligonucleotides, other oligomers (e.g., peptide nucleic acids) or nonpolymeric organic compounds.
In a particularly preferred embodiment, the reagent is prepared by the polymerization of one or more functional group-containing monomers of the formula: 
wherein R1 represents hydrogen or C1-C6 alkyls, and wherein R2 and R3, independently among them, represent hydrogen, C1-C6 alkyls or phenyls containing one or more reactive substituents selected from 
OR5, or SR5 (where R4 is a C1-C6 alkyl or a heterocyclic ring containing one or more nitrogen atoms and R5 is a C1-C6 alkyl or phenyl containing one or more reactive substituents selected from 
Certain monomers of this type are described, for instance, in Righetti (""832), the disclosure of which is incorporated herein by reference. In one preferred embodiment of the present invention, the polymeric reagent is prepared from monomers that include N-acryloyl-amino-ethoxy-ethanol (AAEE) a highly hydrophilic monomer which is extremely resistant to hydrolysis (Chiari, Micheletti, Nesi, Fazio, Righetti; Electrophoresis 15, 1994, 177-186). Other monomers of this type are described in U.S. Pat. No. 5,858,653 the disclosure of which is incorporated herein by reference.
The method and reagent of this invention find particular utility in situations in which it is desired to increase the synthesis capacity without necessarily requiring a corresponding or undue increase in reaction volume. The reagent of the present invention provides a preformed polymer composition in which the polymer molecules can be purified, characterized, and controlled in a manner not heretofore possible.
In a preferred embodiment, the reagent includes the attachment of preformed synthetic polymers to a surface (as distinguished from those formed by polymerization in situ upon the support), and more preferably, the attachment of the preformed polymers by photochemical means. It is also preferred that the functional groups be present at a plurality of positions along the polymer backbone. The number (or average number) and position of functional groups can be controlled by the choice of monomers used to form the polymer, e.g., by the ratio of functional group-containing monomers to xe2x80x9cdiluentxe2x80x9d monomers.
A polymer reagent composition of this invention provides an optimal combination of such properties as swellability, functional group density, reactivity, permeability, hydrophilicity, and hydrolytic stability. In a particularly preferred embodiment, the reagent composition comprises a polymeric derivative providing one or more different reactive groups. The reagent composition can be attached to the surface in any suitable manner, and is preferably covalently attached to the surface, more preferably by the use of photoreactive groups.
Suitable support materials include beads, slides, wafers, films, discs and plates (e.g., microwell plates), prepared from such materials as organosilane-treated glass, organosilane-treated silicon, polypropylene, polyethylene, and polystyrene (optionally cross-linked with divinylbenzene). Additional support materials include grafted polyacrylamide beads, latex beads, dimethylacrylamide beads (optionally cross-linked with N,Nxe2x80x2-bis-acryloyl ethylene diamine), glass particles coated with hydrophobic polymers, etc., (i.e., having a rigid or semi-rigid surface). Divinylbenzene-crosslinked, polyethyleneglycol-grafted polystyrene type beads can be used as well.
In a particularly preferred embodiment, the reagent comprises an hydroxyl-substituted polyacrylamide reagent. Such a reagent can be attached to a surface, e.g., photochemically, in any desired manner and concentration, in order to provide the surface with a desired density of reactive (e.g., primary hydroxyl) groups.
A polymer of this invention can be prepared using any suitable means, e.g., by the reaction of monomers providing one or more functional groups with one or more reactive comonomers (e.g., monomers providing a photoreactive group) and/or with one or more non-reactive comonomers (e.g., xe2x80x9cdiluentxe2x80x9d monomers lacking either a photoreactive group or functional group). Those skilled in the relevant art, given the present description, will appreciate the manner in which a polymer of this invention can be synthesized by free radical polymerization using concentrations and ratios of monomers tailored to achieve the desired surface characteristics. Thus the relative and absolute concentrations of functional groups, as well as the molecular weight of the polymer (and extent of branching, etc.), and the means of immobilizing the polymer (such as by the numbers and/or locations of photoactivatable groups along its length) can all be adjusted to optimize performance.
Comonomers having functional groups of varying types and reactivities, can be selected as well. Although not the only determining factor, the length of whatever spacer may be included between a functional groups and the ultimate polymer backbone can have a predictable or determinable effect on the reactivity of the functional group. In addition, relatively inert monomers can be included, in effect as diluent monomers, in order to adjust the density of the functional groups to desired levels and to achieve the desired polymer characteristics, e.g., to adjust its hydrophilic, hydrophobic, or amphiphilic nature, which in turn can affect its solvation characteristics.
Finally, comonomers can also be included that provide reactive groups for immobilizing the polymer onto a surface. Such monomers preferably contain photoactivatable groups, or can include thermochemically reactive groups that can be used to either attach the polymer directly to a corresponding reactive site or group on the surface, or to another reagent that itself provides a photoactivatable group. For example, hydroxyl groups can be activated with a variety of activating agents (e.g., 1,1-carbonyldiimidazole, 2,2,2-trifluoroethanesulfonyl chloride, or 2-fluoro-1-methylpyridinium p-toluenesulfonate). Such reactions can be used to immobilize a hydroxyl polymer onto a surface containing amine groups (e.g., glass coated with 3-aminopropyltriethoxysilane). In this example, any excess activated hydroxyl groups that do not react with amines on the surface can be hydrolyzed back to free hydroxyl groups. The comonomers can also be selected having different polymerization rates, to optimize the distribution of comomomers in the polymer. Optionally, or in addition, comonomer distribution can be controlled and affected by the preparation and use of block copolymers.
Hydrophilic or amphiphilic polymers are also provided, having means for immobilizing to a surface via stable covalent bonds and multiple functional groups of the type described herein. Such polymers find particular use for solid phase synthesis of peptides, oligonucleotides, similar type polymers (e.g., peptide nucleic acids) and nonpolymeric organic compounds. The use of presynthesized polymers, e.g., as opposed to grafted polymers or those formed in situ, provides a number of advantages, including the ability to purify and characterize the polymer before immobilization.
In yet another aspect, the invention provides a method of providing reactive groups upon a surface, the method including the step of coating the surface with a reagent composition as described herein. In further aspects, the invention provides a support surface coated with such a reagent composition.
In a preferred embodiment, a reagent of this invention is prepared by the polymerization of monomers containing functional groups, optionally and preferably, in combination with other monomers, such as those containing other useful groups, diluent monomers and the like.
In a preferred embodiment, a polymer of the present invention is prepared by polymerizing one or more monomers selected from the group: 
wherein R1 represents hydrogen or C1-C6 alkyls, and wherein R2 and R3, independently among them, represent hydrogen, C1-C6 alkyls or phenyls containing one or more reactive substituents selected from 
OR5, or SR5 (where R4 is a C1-C6 alkyl or a heterocyclic ring containing one or more nitrogen atoms and R5 is a C1-C6 alkyl or phenyl containing one or more reactive substituents selected from 
Optionally, and preferably, the resultant polymers are also attached to the surface via photochemical means, e.g., by the incorporation of one or more photogroups into the polymer by means of photogroup-containing copolymerizable monomers.
Comonomers can be selected to provide any desired property or function, including any desired reactivity. Although not the only determining factor, the length of the spacer between the functional groups and the polymer backbone often has an effect on the reactivity of the functional groups.
In addition, xe2x80x9cinertxe2x80x9d or xe2x80x9cdiluentxe2x80x9d monomers can be used to adjust the density of functional groups to optimal levels and to achieve the desired polymer characteristics, such as hydrophilic or amphiphilic polymers, in order to achieve optimal solvation characteristics. Examples of such monomers include, for instance, acrylamide, N-vinyl pyrrolidone, methacrylamide, N-isopropylacrylamide, N-vinylpyridine, N-vinyl caprolactam, styrene, vinyl acetate, and N-acryloylmorpholine.
A preferred composition of this invention includes one or more pendent latent reactive (preferably photoreactive) groups covalently attached, or adapted to be attached, directly or indirectly, to a copolymerizable monomer. Photoreactive groups are defined herein, and preferred groups are sufficiently stable to be stored under conditions in which they retain such properties. See, e.g., U.S. Pat. No. 5,002,582, the disclosure of which is incorporated herein by reference. Latent reactive groups can be chosen that are responsive to various portions of the electromagnetic spectrum, with those responsive to ultraviolet and visible portions of the spectrum (referred to herein as xe2x80x9cphotoreactivexe2x80x9d) being particularly preferred.
Photoreactive groups respond to specific applied external stimuli to undergo active specie generation with resultant covalent bonding to an adjacent chemical structure, e.g., as provided by the same or a different molecule. Photoreactive groups are those groups of atoms in a molecule that retain their covalent bonds unchanged under conditions of storage but that, upon activation by an external energy source, form covalent bonds with other molecules.
The photoreactive groups generate active species such as free radicals and particularly nitrenes, carbenes, and excited states of ketones upon absorption of electromagnetic energy. Photoreactive groups may be chosen to be responsive to various portions of the electromagnetic spectrum, and photoreactive groups that are responsive to e.g., ultraviolet and visible portions of the spectrum are preferred and may be referred to herein occasionally as xe2x80x9cphotochemical groupxe2x80x9d or xe2x80x9cphotogroupxe2x80x9d.
Photoreactive aryl ketones are preferred, such as acetophenone, benzophenone, anthraquinone, anthrone, and anthrone-like heterocycles (i.e., heterocyclic analogs of anthrone such as those having N, O, or S in the 10-position), or their substituted (e.g., ring substituted) derivatives. Examples of preferred aryl ketones include heterocyclic derivatives of anthrone, including acridone, xanthone, and thioxanthone, and their ring substituted derivatives.
The functional groups of such ketones are preferred since they are readily capable of undergoing the activation/inactivation/reactivation cycle described herein. Benzophenone is a particularly preferred photoreactive moiety, since it is capable of photochemical excitation with the initial formation of an excited singlet state that undergoes intersystem crossing to the triplet state. The excited triplet state can insert into carbon-hydrogen bonds by abstraction of a hydrogen atom (from a support surface, for example), thus creating a radical pair. Subsequent collapse of the radical pair leads to formation of a new carbon-carbon bond. If a reactive bond (e.g., carbon-hydrogen) is not available for bonding, the ultraviolet light-induced excitation of the benzophenone group is reversible and the molecule returns to ground state energy level upon removal of the energy source. Photoactivatible aryl ketones such as benzophenone and acetophenone are of particular importance inasmuch as these groups are subject to multiple reactivation in water and hence provide increased coating efficiency.
The azides constitute an additional preferred class of photoreactive groups and include arylazides (C6R5N3) such as phenyl azide and particularly 4-fluoro-3-nitrophenyl azide, acyl azides (xe2x80x94COxe2x80x94N3) such as benzoyl azide and p-methylbenzoyl azide, azido formates (xe2x80x94Oxe2x80x94COxe2x80x94N3) such as ethyl azidoformate, phenyl azidoformate, sulfonyl azides (xe2x80x94SO2xe2x80x94N3) such as benzenesulfonyl azide, and phosphoryl azides (RO)2PON3 such as diphenyl phosphoryl azide and diethyl phosphoryl azide. Diazo compounds constitute another class of photoreactive groups and include diazoalkanes (xe2x80x94CHN2) such as diazomethane and diphenyldiazomethane, diazoketones (xe2x80x94COxe2x80x94CHN2) such as diazoacetophenone and 1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (xe2x80x94Oxe2x80x94COxe2x80x94CHN2) such as t-butyl diazoacetate and phenyl diazoacetate, and beta-keto-alpha-diazoacetates (xe2x80x94COxe2x80x94CN2xe2x80x94COxe2x80x94Oxe2x80x94) such as t-butyl alpha diazoacetoacetate. Other photoreactive groups include the diazirines (xe2x80x94CHN2) such as 3-trifluoromethyl-3-phenyldiazirine, and ketenes (xe2x80x94CHxe2x95x90Cxe2x95x90O) such as ketene and diphenylketene.
Upon activation of the photoreactive groups, the reagent molecules are covalently bound to each other and/or to the material surface by covalent bonds through residues of the photoreactive groups. Exemplary photoreactive groups, and their residues upon activation, are shown as follows (wherein R and Rxe2x80x2 can be any non-interfering organic groups).
The photoactivatable monomers of the invention can be applied to any surface having carbon-hydrogen bonds, with which the photoreactive groups can react to immobilize the resulting polyacrylamide to surfaces. Examples of appropriate substrates include, but are not limited to, polypropylene, polystyrene, poly(vinyl chloride), polycarbonate, poly(methyl methacrylate), parylene and any of the numerous organosilanes used to pretreat glass or other inorganic surfaces.
Polymers of this invention are preferably synthesized by free radical polymerization using concentrations and ratios of monomers that are tailored to achieve the desired surface characteristics. Thus the levels of functional groups, the molecular weight of the polymer and the means of immobilizing the polymer (e.g., by the incorporation of photoactivatable groups), can be adjusted by those skilled in the art to achieve any desired product and/or to optimize the performance or physical-chemical characteristics in one or more respects.
A reagent of the present invention can be used in a variety of ways to provide functionalized support surfaces for use in solid phase synthesis. In one embodiment, the reagent can be packaged and provided separately, and optionally in bulk, to be applied to a surface by the user at the time of use. In another embodiment, the reagent can be applied and covalently bound to a support (e.g., by photochemical means) at the time of manufacturing the support itself, and the resultant coated support material can be packaged and sold in a form substantially ready for use.
The following Examples are provided to illustrate, but not limit the present invention. While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims.